With completion of the genome sequence and the estimated 20-25,000 genes comprising its entirety, it has become clear to investigators that the complexity of humans cannot be fully explained by so few genes. Alternative splicing of mRNA provides an intricate means by which individual genes can be expressed in various isoforms and it is through this process that each mRNA can be translated into several different proteins. This complexity thus provides a rich source for identifying differentially expressed gene isoforms and thus, potential diagnostic markers. Because the clinician and surgeon cannot determine malignancy pre- or intra-operatively, patients with indeterminate thyroid lesions on fine needle aspiration (FNA) cannot be optimally clinically managed. Therefore, additional diagnostic markers of malignancy are greatly needed. We therefore propose to examine thyroid tumors by splice array analysis for differentially expressed alternative splice variants, validate our findings by RTPCR, in situ hybridization, or immunohistochemistry and test the applicability of our assay on FNA biopsies form thyroid nodules that are indeterminate diagnostically. The ability to distinguish benign from malignant thyroid tumors using this novel molecular approach could improve both the clinical and surgical management of over 100,000 new patients in the United States annually who present with indeterminate or inadequate thyroid FNA samples. Indeed, potential application of this assay could dramatically improve the clinical management of these patients. PUBLIC HEALTH RELEVANCE: 350,000 fine needle aspirations (FNA) are performed annually in the United States to diagnose thyroid nodules, and of these, over 100,000 are indeterminate or inadequate for diagnosis. We therefore will study these tumors for differentiating molecular markers. For patients with these FNA diagnoses, use of differentiating molecular patterns could dramatically improve their surgical management by obviating the need for two operations in the case of a malignancy or, by obviating the need a patient undergoing any surgery in the case of a benign tumor.